Nuclear magnetic resonance (NMR) spectrometers typically include a superconducting magnet for generating a static magnetic field B0, and an NMR probe including one or more special-purpose radio-frequency (RF) coils for generating a time-varying magnetic field B1 perpendicular to the field B0, and for detecting the response of a sample to the applied magnetic fields. Each RF together with its associated circuitry can resonate at the Larmor frequency of a nucleus of interest present in the sample. The RF coils are typically provided as part of an NMR probe, and are used to analyze samples situated in sample tubes or flow cells.
An NMR frequency of interest is determined by the nucleus of interest and the strength of the applied static magnetic field B0. In order to maximize the sensitivity of NMR measurements, the resonant frequency of the excitation/detection circuitry is set to be equal to the frequency of interest. The resonant frequency of the excitation/detection circuitry varies asν=½π√{square root over (LC)}  [1]where L and C are the effective inductance and capacitance, respectively, of the excitation/detection circuitry.
The chemical shift of a nucleus is the normalized difference between the resonance frequency of the nucleus and a standard, normalized to the standard. Chemical shifts expressed in ppm may be determined according to the relation:
                    δ        =                                            (                              υ                -                                  υ                  REF                                            )                        ×                          10              6                                            υ            REF                                              [        2        ]            wherein υREF is the resonance frequency of a reference (standard) nucleus. Common standards employed in NMR spectroscopy include tetramethylsilane (TMS) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), among others. The reference (standard) resonance frequency is often measured by adding the standard compound (e.g. TMS) to the sample of interest.
For many applications, it may be impractical to add a standard compound to the sample of interest. For example, adding a standard compound may be unacceptable for some biological samples, or for samples that include molecules that react with the standard or whose reactivity with the standard is unknown. Other experiments cannot tolerate any additive, such as IC50 measurements requiring samples to be analyzed for subsequent in-vivo applications.
In the article “Concentration Measurement by Proton NMR using the ERETIC Method,” Analytical Chemistry 71(13): 2554-2557, July 1999, Akoka et al. describe a method, named ERETIC (Electronic REference To access In vivo Concentrations), in which a reference signal is synthesized by an electronic device. Akoka et al. also describe NMR synthetic reference systems and methods in French Patent Publication No. 2 735 865 A1. Known NMR systems and methods employing electronically-synthesized reference signals commonly employ a spare probe channel to couple the reference signals into the observe channel, and may allow limited control over system parameters.